Ectodysplasin (Eda) plays important roles in both shaping the developing tooth and establishing the number of teeth within the tooth row. Sonic hedgehog (Shh) has been shown to act downstream of Eda and is involved in the initiation of tooth development. Eda-/- mice possess hypoplastic and hypomineralized incisors and show changes in tooth number in the molar region. In the present study we used 3D reconstruction combined with expression analysis, cell lineage tracing experiments, and western blot analysis in order to investigate the formation of the incisor germs in Eda-/- mice. We show that a lack of functional Eda protein during early stages of incisor tooth germ development had minimal impact on development of the early expression of Shh in the incisor, a region proposed to mark formation of a rudimental incisor placode and act as an initiating signalling centre. In contrast, deficiency of Eda protein had a later impact on expression of Shh in the primary enamel knot of the functional tooth. Eda-/- mice had a smaller region where Shh was expressed, and a reduced contribution from Shh descendant cells. The reduction in the enamel knot led to the formation of an abnormal enamel organ creating a hypoplastic functional incisor. Eda therefore appears to influence the spatial formation of the successional signalling centres during odontogenesis.

1st Department of Medicine Department of Hematology 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czechia

Department of Cell Biology Faculty of Science Charles University Prague Czechia

Department of Craniofacial and Regenerative Biology King´s College London Guys Hospital London United Kingdom

Department of Histology and Embryology 3rd Faculty of Medicine Charles University Prague Czechia

Department of Radiation Dosimetry Nuclear Physics Institute Czech Academy of Sciences Prague Czechia

Institute of Histology and Embryology 1st Faculty of Medicine Charles University Prague Czechia

Laboratory of Odontogenesis and Osteogenesis Institute of Animal Physiology and Genetics Academy of Sciences Brno Czechia

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Ahtiainen L., Uski I., Thesleff I., Mikkola M. L. (2016). Early epithelial signaling center governs tooth budding morphogenesis. Journal of Cell Biology. 12 (6), 753–767. 10.1083/jcb.201512074 PubMed DOI PMC

Arte S., Parmanen S., Pirinen S., Alaluusua S., Nieminen P. (2013). Candidate gene analysis of tooth agenesis identifies novel mutations in six genes and suggests significant role for WNT and EDA signaling and allele combinations. PLoS One 22 (8), e73705. 10.1371/journal.pone.0073705 PubMed DOI PMC

Baka S., Malamitsi-Puchner A., Boutsikou T., Boutsikou M., Marmarinos A., Hassiakos D., et al. (2015). Cord blood irisin at the extremes of fetal growth. Metabolism 64 (11), 1515–1520. 10.1016/j.metabol.2015.07.020 PubMed DOI

Bitgood M. J., McMahon A. P. (1995). Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Developmental Biology 172 (1), 126–138. 10.1006/dbio.1995.0010 PubMed DOI

Blecher S. R. (1986). Anhidrosis and absence of sweat glands in mice hemizygous for the tabby gene: Supportive evidence for the hypothesis of homology between tabby and human anhidrotic (hypohidrotic) ectodermal dysplasia (Christ-Siemens-Touraine syndrome). Journal of Investigative Dermatologyl 87 (6), 720–722. 10.1111/1523-1747.ep12456718 PubMed DOI

Butler P. M. (1956). The ontogeny of molar pattern. Biological Reviews of the Cambridge Philosophical Society 31, 30–69. 10.1111/j.1469-185X.1956.tb01551.x DOI

Cobourne M. T., Sharpe P. T. (2010). Making up the numbers: The molecular control of mammalian dental formula. Seminars in Cell & Developmental Biology. 21 (3), 314–324. 10.1016/j.semcdb.2010.01.007 PubMed DOI

Cui C. Y., Schlessinger D. (2006). EDA signaling and skin appendage development. Cell Cycle 5 (21), 2477–2483. 10.4161/cc.5.21.3403 PubMed DOI PMC

Cui C. Y., Yin M., Sima J., Childress V., Michel M., Piao Y., et al. (2014). Involvement of Wnt, Eda and Shh at defined stages of sweat gland development. Development 141 (19), 3752–3760. 10.1242/dev.109231 PubMed DOI PMC

Dassule H. R., Lewis P., Bei M., Maas R., McMahon A. P. (2000). Sonic hedgehog regulates growth and morphogenesis of the tooth. Development. 127 (22), 4775–4785. 10.1242/dev.127.22.4775 PubMed DOI

Fons Romero J. M., Star H., Lav R., Watkins S., Harrison M., Hovorakova M., et al. (2017). The impact of the Eda pathway on tooth root development. Journal of Dental Research. 96 (11), 1290–1297. 10.1177/0022034517725692 PubMed DOI PMC

Grüneberg H. (1965). Genes and genotypes affecting the teeth of the mouse. Journal of Embryology and Experimental Morphology. 14 (2), 137–159. 10.1242/dev.14.2.137 PubMed DOI

Hardcastle Z., Mo R., Hui C. C., Sharpe P. T. (1998). The Shh signalling pathway in tooth development: Defects in Gli2 and Gli3 mutants. Development. 125 (15), 2803–2811. 10.1242/dev.125.15.2803 PubMed DOI

Harfe B. D., Scherz P. J., Nissim S., Tian H., McMahon A. P., Tabin C. J. (2004). Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118 (4), 517–528. 10.1016/j.cell.2004.07.024 PubMed DOI

Hayashi S., McMahon A. P. (2002). Efficient recombination in diverse tissues by a tamoxifen-inducible form of cre: A tool for temporally regulated gene activation/inactivation in the mouse. Developmental Biology. 244 (2), 305–318. 10.1006/dbio.2002.05971006 PubMed DOI

Hovorakova M., Lochovska K., Zahradnicek O., Domonkosova Tibenska K., Dornhoferova M., Horakova-Smrckova L., et al. (2016). One odontogenic cell-population contributes to the development of the mouse incisors and of the oral vestibule. PLoS One 11 (9), e0162523. 10.1371/journal.pone.0162523 PubMed DOI PMC

Hovorakova M., Prochazka J., Lesot H., Smrckova L., Churava S., Boran T., et al. (2011). Shh expression in a rudimentary tooth offers new insights into development of the mouse incisor. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution. 316 (5), 347–358. 10.1002/jez.b.21408 PubMed DOI

Hovorakova M., Smrckova L., Lesot H., Lochovska K., Peterka M., Peterkova R. (2013). Sequential Shh expression in the development of the mouse upper functional incisor. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution. 320 (7), 455–464. 10.1002/jez.b.22522 PubMed DOI

Iseki S., Araga A., Ohuchi H., Nohno T., Yoshioka H., Hayashi F., et al. (1996). Sonic hedgehog is expressed in epithelial cells during development of whisker, hair, and tooth. Biochem. Biophys. Res. Commun. 218 (3), 688–93. 10.1006/bbrc.1996.0123 PubMed DOI

Jernvall J., Kettunen P., Karavanova I., Martin L. B., Thesleff I. (1994). Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: Non-dividing cells express growth stimulating fgf-4 gene. The International Journal of Developmental Biology. 38 (3), 463–469. 10.1387/IJDB.7848830 PubMed DOI

Kere J., Srivastava A. K., Montonen O., Zonana J., Thomas N., Ferguson B., et al. (1996). X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein. Nature Genetics. 13 (4), 409–416. 10.1038/ng0895-409 PubMed DOI

Laurikkala J., Mikkola M., Mustonen T., Aberg T., Koppinen P., Pispa J., et al. (2001). TNF signaling via the ligand-receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis. Developmental Biology. 229 (2), 443–455. 10.1006/dbio.2000.9955 PubMed DOI

Lefebvre S., Mikkola M. L. (2014). Ectodysplasin research–where to next? Semin immunol. Seminars in Immunology. 26 (3), 220–228. Review. 10.1016/j.smim.2014.05.002 PubMed DOI

Lesot H., Vonesch J. L., Peterka M., Turecková J., Peterková R., Ruch J. V. (1996). Mouse molar morphogenesis revisited by three-dimensional reconstruction. II. Spatial distribution of mitoses and apoptosis in cap to bell staged first and second upper molar teeth. The International Journal of Developmental Biology. 40 (5), 1017–1031. PubMed

Lesot H., Kristenova P., Haze A., Deutsch D., Peterka M., Peterkova R. (2004). Lower Incisors in the Tabby Mouse, 8th International Conference on the Chemistry and Biology of Mineralized Tissues (ICCBMT), Banff, Alberta, Canada. 2004, 196–199. 10.13140/RG.2.1.3144.6568 DOI

Li J., Chatzeli L., Panousopoulou E., Tucker A. S., Green J. B. (2016). Epithelial stratification and placode invagination are separable functions in early morphogenesis of the molar tooth. Development 143 (4), 670–681. 10.1242/dev.130187 PubMed DOI PMC

Miard S., Peterková R., Vonesch J. L., Peterka M., Ruch J. V., Lesot H. (1999). Alterations in the incisor development in the Tabby mouse. The International Journal of Developmental Biology. 43 (6), 517–529. PubMed

Mikkola M. L. (2008). TNF superfamily in skin appendage development. Cytokine & Growth Factor Reviews. 19 (3-4), 219–230. Review. 10.1016/j.cytogfr.2008.04.008 PubMed DOI

Mikkola M. L., Thesleff I. (2003). Ectodysplasin signaling in development. Cytokine & Growth Factor Reviews. 14 (3-4), 211–224. 10.1016/s1359-6101(03)00020-0 PubMed DOI

Mustonen T., Ilmonen M., Pummila M., Kangas A. T., Laurikkala J., Jaatinen R., et al. (2004). Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages. Development 131 (20), 4907–4919. 10.1242/dev.01377 PubMed DOI

Peterka M., Lesot H., Peterková R. (2002). Body weight in mouse embryos specifies staging of tooth development. Connective Tissue Research. 43 (2-3), 186–190. 10.1080/03008200290000673 PubMed DOI

Peterková R., Kristenová P., Lesot H., Lisi S., Vonesch J. L., Gendrault J. L., et al. (2002). Different morphotypes of the tabby (EDA) dentition in the mouse mandible result from a defect in the mesio-distal segmentation of dental epithelium. Orthodontics & Craniofacial Research. 5 (4), 215–226. 10.1034/j.1600-0544.2002.02226.x PubMed DOI

Pispa J., Jung H. S., Jernvall J., Kettunen P., Mustonen T., Tabata M. J., et al. (1999). Cusp patterning defect in Tabby mouse teeth and its partial rescue by FGF. Developmental Biology. 216 (2), 521–534. 10.1006/dbio.1999.9514 PubMed DOI

Risnes S., Peterkova R., Lesot H. (2005). Distribution and structure of dental enamel in incisors of Tabby mice. Archives of Oral Biology. 50 (2), 181–184. 10.1016/j.archoralbio.2004.11.003 PubMed DOI

Sehic A., Peterkova R., Lesot H., Risnes S. (2009). Distribution and structure of the initial dental enamel formed in incisors of young wild-type and Tabby mice. European Journal of Oral Sciences. 117 (6), 644–654. 10.1111/j.1600-0722.2009.00676.x PubMed DOI

Srivastava A. K., Pispa J., Hartung A. J., Du Y., Ezer S., Jenks T., et al. (1997). The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains. Proceedings of the National Academy of Sciences of the United States of America. 94 (24), 13069–13074. 10.1073/pnas.94.24.13069 PubMed DOI PMC

Tucker A. S., Headon D. J., Schneider P., Ferguson B. M., Overbeek P., Tschopp J., et al. (2000). Edar/Eda interactions regulate enamel knot formation in tooth morphogenesis. Development 127 (21), 4691–4700. 10.1242/dev.127.21.4691 PubMed DOI

Wang F., Du M., Wang R., Zhou J., Zhang W., Li H. (2015). Molecular mechanism of Hoxd13-mediated congenital malformations in rat embryos. International Journal of Clinical and Experimental Pathology. 8 (12), 15591–15598. PubMed PMC

Weeks N. L., Blecher S. R. (1983). Evidence from thiol histochemistry for homology between the Tabby-crinkled syndrome in mice and human ectodermal dysplasia. Journal of Histochemistry and Cytochemistry. 31 (12), 1407–1411. 10.1177/31.12.6631002 PubMed DOI

Zhang X., Zhou X., Li L., Sun M., Gao Q., Zhang P., et al. (2016). Chronic hypoxia in pregnancy affects thymus development in Balb/c mouse offspring via IL2 Signaling. Molecular Reproduction and Development. 83 (4), 337–346. 10.1002/mrd.22630 PubMed DOI